Trapping interactions between catalytic domains and carrier proteins of modular biosynthetic enzymes with chemical probes

Abstract

Covering: up to early 2018 The Nonribosomal Peptide Synthetases (NRPSs) and Polyketide Synthases (PKSs) are families of modular enzymes that produce a tremendous diversity of natural products, with antibacterial, antifungal, immunosuppressive, and anticancer activities. Both enzymes utilize a fascinating modular architecture in which the synthetic intermediates are covalently attached to a peptidyl- or acyl-carrier protein that is delivered to catalytic domains for natural product elongation, modification, and termination. An investigation of the structural mechanism therefore requires trapping the often transient interactions between the carrier and catalytic domains. Many novel chemical probes have been produced to enable the structural and functional investigation of multidomain NRPS and PKS structures. This review will describe the design and implementation of the chemical tools that have proven to be useful in biochemical and biophysical studies of these natural product biosynthetic enzymes.

Mechanistic Probes for the Epimerization Domain of Nonribosomal Peptide Synthetases

“…Central in mediating NRP synthesis is the peptidyl carrier protein (PCP), which acts as a scaffold, tethering amino acid building blocks and peptidyl intermediates through a 4′‐phosphopantetheine prosthetic arm as they are modified and condensed by other domains in the module . The past decade has seen remarkable progress in the structural and mechanistic elucidation of NRPS domains through the use of biochemical, structural, and genetic methods . Despite these achievements, new strategies to explore the enzymology of these complex, natural product–producing assembly lines are in constant development.…”

Dynamic visualization of type II peptidyl carrier protein recognition in pyoluteorin biosynthesis

“…Carrier proteins (CPs) and partner proteins often form weak transient interactions and high disassociation may impede co-crystallization. 12 To stabilize the interaction between PltL and PltF, a substrate mimic of the proline adenosine monophosphate (Pro-AMP) intermediate was deemed necessary. Based on a covalent inhibitor motif developed by the Aldrich and Tan groups, [13][14][15][16][17][18] the proline adenosine vinylsulfonamide (Pro-AVSN) was synthesized (see ESI †) and employed to trap PltL with PltF ( Fig.…”

Structure and Dynamic Basis of Molecular Recognition Between Acyltransferase and Carrier Protein inE. coliFatty Acid Synthesis

“…Classically, trapping covalent complexes between an ACP and a partner enzyme (PE) has been achieved through the synthesis of pantetheinamide crosslinking probes that react selectively and uniformly with the active site residues of the PE. [42][43][44] These probes can be loaded onto an ACP via a one-pot chemoenzymatic method 45 to produce crypto-ACPs, which, when mixed with cognate PEs, form crosslinked complexes. [46][47][48][49] Despite recent successes developing active site fluorescent probes for ATs, 50 targeting the active site serine of FabD with complementary pantetheinamide probes has proved challenging.…”